Idiopathic pulmonary fibrosis (IPF) is known as a progressive, chronic, and irreversible lung disease that has symptoms such as the irreversible decline in lung function, progressive pulmonary scarring, and common interstitial pneumonia (Ding et al., 2015; Fanny et al., 2018; Kamio et al., 2018). IPF has effects on more than 3 million people worldwide. The global annual incidence of IPF is 2- 9 people per 100, 000 (Hutchinson et al., 2015) and the median survival time is solely 2–3 years after diagnosis is achieved (Ohta et al., 2017). Previous studies have shown that the risk factors of IPF include genetic factors, smoking, airway inflammation, occupational exposure, etc(H. Y. Lee et al., 2020). Basically, it is currently accepted that the development and progression of pulmonary fibrosis are attributable to aberrant repair following repeated alveolar epithelial cell injury in response to different stimuli. However, its exact pathogenesis is not clear yet (Wolters et al., 2018; Wu et al., 2020).

Recently, an iron-dependent and non-apoptotic regulatory cell death mechanism have been discovered and named ferroptosis (Zhang et al., 2021). Ferroptosis is widely involved in the development of stroke, brain injury, and tumors (Bu et al., 2021; Guan et al., 2021; Hu et al., 2021). However, iron is an essential reagent for normal physiological activities of cells, ferroptosis is distinguished from other forms of cell death such as apoptosis, necrosis, and autophagy by the lethal iron-dependent accumulation of lipid-based reactive oxygen species (ROS) (Ye et al., 2019). The reason is that the excessive accumulation of iron in cells causes lipid peroxidation elevation leading to this type of cell death (Nishizawa et al., 2021). Notably, ferroptosis-related heterozygous gene GPX4 flawed mice showed more severe bleomycin-induced pulmonary fibrosis, while this effect was attenuated in transgenic mice (Tsubouchi et al., 2019), indicating that ferroptosis may be substantially contributing to the pulmonary fibrosis. In addition, Yatmark et al. (2015) showed that excessive iron load in the lung would lead to pulmonary fibrosis and alveolar epithelial cell damage, and a ferrous ion chelator such as deferoxamine (DFO) can reduce the degree of excessive iron-induced lung injury. Therefore, further investigations are required to detect the ferroptosis-related genes including those involved in the pulmonary fibrosis that is largely unknown at present. Since probing and discovering the underlying mechanism of ferroptosis involvement in pulmonary fibrosis can be a potential therapeutical target for its treatment.

Currently, to the knowledge of authors, no bioinformatic-based study has targeted the mechanism underlying ferroptosis genes’ contribution to pulmonary fibrosis. Therefore, in the present study, data analysis and data mining techniques were used to screen the differentially expressed genes (DEGs) in IPF patients compared to the normal lung tissues. Then, the identified DEGs were intersected with the ferroptosis data set, and the random forest algorithm was used to achieve the key ferroptosis DEGs. We also created mice models of pulmonary fibrosis using bleomycin-induced mice and tested these hypotheses through a treadmill training intervention. Our results will contribute to the understanding of the ferroptosis mechanism in pulmonary fibrosis as well as providing new ideas for IFP clinical diagnosis and its treatment.

Ethics Committee of the First Affiliated Hospital of Chengdu Medical College approved all procedures (Approval No. 2021CYFYIRB-BA-32-01). The animal experiments involved in the research content and process of the project met the ethical requirements of experimental animal welfare and complied with the Animal Protection Law and relevant regulations. The environmental conditions of animal laboratory facilities were in line with the Chinese national standard “Experimental Animals and Environmental Facilities” (GB14925-2010), and the animal feeding management and animal experiment operation were in line with the requirements of the regulations of Chengdu Medical College on the management of experimental animals.

The present study identified the key ferroptosis-related genes in pulmonary fibrosis and explored the potential pathogenesis of ferroptosis in pulmonary fibrosis. Here, a total of 20 DEGs were obtained from the cross set of GSE150910 and FerrDb datasets, including and 13 upregulated genes and 7 downregulated genes with significant interactions among them. Then, the results of enrichment analysis using R software indicated that these genes were mainly involved in the HIF1-α signaling pathway and hypoxia response. Therefore, we used treadmill training to verify the pulmonary fibrosis mice model. Animal experiments showed that exercise might affect the expression of ferroptosis-related genes and inhibit ferroptosis in pulmonary fibrosis confirming the reliability of bioinformatics analysis. Additionally, this project achieved several genes that have not been previously related to ferroptosis and/or pulmonary fibrosis. From a bioinformatics perspective, the present study has provided a substantial reference for the potential mechanisms of pulmonary fibrosis pathogenesis.

In general, the excess accumulation of iron, the lipid-based intracellular ROS overaccumulation, and eventually the lipid oxidation are the main characterizations of ferroptosis which cause damages to the cell membrane leading to cell death (Honarpisheh et al., 2016). This type of cell death often occurs due to the oxidative stress caused by the excess ROS mainly accumulated during the aerobic metabolisms (Ning et al., 2017). Our enrichment analyses showed that after such oxidative stresses, some signaling pathways such as the HIF1-α signaling pathway are activated that can lead to ferroptosis or cell apoptosis through inducing free radicals’ generation after pulmonary fibrosis (Epstein Shochet et al., 2021). The application of antioxidants for treating pulmonary fibrosis has been widely studied (Venkatadri et al., 2017), one reason of which, can be their potential of reducing ferroptosis during pulmonary fibrosis. It has been previously confirmed that iron overaccumulation can activate the HIF1-α signaling pathway. Therefore, it is hypothesized that inhibiting this activation process can reduce the intracellular content of iron and ameliorate the alveolar epithelial cell function (Speer et al., 2013; Xu et al., 2017). In consistence with our bioinformatics analysis, these results suggest that the HIF1-α signaling pathway may be activated by ferroptosis, promote ROS production, and aggravate cell damage, leading to a vicious cycle.

Furthermore, we obtained a module composed of 10 ferroptosis-related genes (CAV1, JUN, NOS2, EPAS1, GDF15, SLC2A1, CDKN2A, CA9, HNF4A, TP63) using PPI analysis. These genes received a random forests sequencing, and the top five genes were screened as CAV1, NOS2, GDF15, HNF4A, CDKN2A, respectively. Random forest algorithm is a classifier in machine learning. It has good adaptability to complex data and can sort various variables with high prediction accuracy and improve test efficiency (Liu et al., 2021). Accordingly, our five screened-out ferroptosis-related genes showed high reliability. CAV1 is a membrane-bound scaffold protein family which is related to CAV2 and CAV3 protein families (Lillo Urzúa et al., 2020). CAV1 is widely expressed in lung tissues including alveolar epithelial cells, endothelial cells, fibroblasts, and smooth muscle cells. Therefore, the CAV1 protein loss can cause a variety of respiratory diseases (Royce and Le Saux, 2014; Fang et al., 2017; Li et al., 2019). Many studies have also discussed that CAV1 expression is significantly reduced in lung tissues of IPF patients, which is consistent with the results of our bioinformatics analysis. The reason is that normal expression of CAV1 can prevent TGF-βR (the growth factor-β receptor) from transforming, and thus, inhibits the overexpression of transforming growth factor (TGF-β). Reduced expression of CAV1 will weaken the inhibitory effect of TGF-βR, thereby, activating the TGF-β signaling pathway, resulting in a large amount of extracellular matrix (ECM) generation, causing the occurrence of pulmonary fibrosis (Lee et al., 2007; He et al., 2015; Budi et al., 2016). In our pulmonary fibrosis mice model, it was further demonstrated that the mRNA expression level of CAV1 in bleomycin-induced pulmonary fibrosis tissues was significantly lower compared to the normal control group. Moreover, the overall survival analysis verified a longer survival time in patients with high CAV1 expression. In the same regard, Lin et al. (2019). indicated that CAV1 is a major component of cellulose that regulates cell signaling and endocytosis. Overexpression of CAV1 can reduce infiltration of neutrophils and monocytes/macrophages and prevent bleomycin-induced pulmonary fibrosis, which may be related to its key regulatory role in inflammatory activities. Interestingly, our animal experiment showed that CAV1 mRNA expression increased when treadmill exercise was performed in mice with pulmonary fibrosis. El-Mafarjeh et al. (El-Mafarjeh et al., 2020) showed that treadmill exercise improves pulmonary and systemic inflammation in bleomycin-induced pulmonary fibrosis models. This may be related to the fact in this study that treadmill increases CAV1, but the specific mechanisms need to be confirmed by further experiments. CDKN2A, known as the cycle-dependent kinase inhibitor gene, belongs to the cycle-dependent kinase inhibitor family and is an important tumor suppressor gene (Llanos et al., 2015). Du et al. (2018). showed that the lower expression level of CDKN2A mRNA in peripheral blood of IPF patients compared to the control group may be related to the activation of the P53 signaling pathway. In the present experiment, the lower expression level of CDKN2A mRNA in the lung tissues of mice with pulmonary fibrosis compared to that of the normal control group was consistent with the results of the a forementioned study. FERDB online tool predicted that CDKN2A was associated with ferroptosis and classified it as ferroptosis driver. The hypothesis is based on Chen’s study (Chen et al., 2017). However, Chen et al. did not directly demonstrate that CDKN2A could play a major role in promoting ferroptosis. Whether CDKN2A is a driver of ferroptosis in IPF had not been defined. Thus, our results suggested that CDKN2A influencing the ferroptosis of pulmonary fibrosis, the mechanism for which warranted further study. NOS2 is a key gene of oxidative metabolism. Previous studies have reported that under pathophysiological conditions of pulmonary fibrosis, an abnormally high concentration of nitric oxide exists, which may be due to the elevated activity of inducible nitric oxide synthase (NOS2). Excessive nitric oxide production may contribute to fibrosis development (Jang et al., 2004). GDF15 is an endocrine hormone and a member of the transforming growth factor β superfamily that promotes ferroptosis in tumor cells (Shao et al., 2021). Zhang (Zhang et al., 2019) et al. reported that GDF15 is a secreted protein of epithelial origin and may be a useful biomarker of epithelial stress since its increased expression associates with a poorer prognosis in IPF patients, which is consistent with the findings of the present bioinformatics analyses in this study. We speculate that GDF15 may promote ferroptosis in pulmonary fibrosis, thus, aggravate the inflammatory responses in lung tissues and accelerate the process of pulmonary fibrosis. HNF4A is a nuclear transcription factor that binds to DNA as a homodimer and mainly relates to fibrosis development in the liver. Knocking down HNF4A has been shown that can alleviate liver fibrosis (Ji et al., 2019). However, no studies have been reported on HNF4A association with pulmonary fibrosis. Similarly, the animal experiments and survival analysis of bioinformatics analysis in this study showed that HNF4A has no association with pulmonary fibrosis. Therefore, the relationship between HNF4A and pulmonary fibrosis remains unclear and needs further verification by fundamental experiment study. Notably, Data mining showed that NOS2 and CAV1 were downregulated, and CDKN2A and GDF15 were upregulated in IPF tissues. However, NOS2 was upregulated and CDKN2A was downregulated in bleomycin-induced mouse IPF tissues. The inconsistency between experimental results and those obtained using bioinformatic analysis may be due to the following reasons. The data used for the bioinformatic analysis was collected on IPF patients, while experimental data derived from bleomycin-induced mice. Although, human and mouse had very high genetic homology, different species may lead to some bias in results. In an addition, some gene expansions might be greatly temporally and spatially specific, which resulted in the discordance of bioinformatic analysis and experimental results. We will collect samples from IPF patients and perform more extensive validation studies in the future.

Nevertheless, our study has some limitations. 1) Obtaining bioinformatics results from lung tissues of IPF patients and normal lung tissues as well as insufficient collection of clinical tissue samples for validation due to experimental conditions and hospital size. 2) Verification of expression levels of different ferroptosis genes in animal models and lack of potential study of hub genes in cellular models of pulmonary fibrosis. Therefore, further verification is required in the future. 3) The mRNA expression values were further standardized by different standardization method, which might yield differing findings. Most of the data to date is restricted to the detection of changes in gene expression and needs to be validated by further functional experiments.

We identified 20 potential ferroptosis-related genes in pulmonary fibrosis by bioinformatics analysis. CAV1, NOS2, GDF15, CDKN2A may influence the development of pulmonary fibrosis by regulating ferroptosis. Aerobic exercise training after induction of pulmonary fibrosis may attenuate ferroptosis in lung. These results may expand our understanding of pulmonary fibrosis and may contribute to the treatment of pulmonary fibrosis.